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Journal of Virology, August 2006, p. 8283-8285, Vol. 80, No. 16
0022-538X/06/$08.00+0     doi:10.1128/JVI.00813-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Fusion of Tags Induces Spurious Phosphorylation of Rotavirus NSP5


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LETTER
 
Several findings in the paper by Sen et al. entitled "Hyperphosphorylation of the rotavirus NSP5 protein is independent of serine 67 or NSP2, and the intrinsic insolubility of NSP5 is regulated by cellular phosphatases" (5) seem to contradict previous published findings from our laboratory. The authors also state that "... hyperphosphorylated NSP5 is predominantly present in previously unrecognized cellular fractions that are insoluble in 0.2% SDS." We were puzzled by the fact that not a single experiment in this paper was performed with the natural protein. In all cases NSP5 was fused with a His6-G, GAL4, or Myc tag, but the conclusions were extended to the behavior of wild-type NSP5 (wtNSP5). Based on previous results showing that the addition of a tag at the amino terminus of NSP5 drastically changes the characteristics of the protein (e.g., viroplasm-like structure formation without NSP2 [4; unpublished results]), we addressed this point by expressing various NSP5 constructs either untagged or N-terminally tagged with the SV5 peptide (3).

As shown in Fig. 1A and in agreement with previous results of this group (2, 3), the soluble fraction of wild-type NSP5 and its mutants NSP5a (Ser63, 65, 67Ala) and NSP5-Ser67Ala were little or not phosphorylated, whereas the mutation of Ser67 into aspartic acid (NSP5-S67D) was associated with hyperphosphorylation of NSP5. In contrast, tagging of NSP5 at the N terminus increased hyperphosphorylation of SV5-NSP5 and SV5-NSP5-S67D, whereas the effect on the mutants SV5-NSP5a and SV5-NSP5-S67A was reduced. Analysis of the insoluble fractions showed that the relative amounts of the nontagged NSP5 constructs were less than 10% of the total and did not show a phosphorylation pattern different from that of the soluble fraction (Fig. 1A). By contrast, N-tagged constructs showed a pattern of increased phosphorylation in the insoluble fraction, yet this represented in all cases not more than 20% of the total. These results demonstrate that there is a substantial difference in the behavior of tagged versus nontagged NSP5 proteins.


Figure 1
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  		FIG. 1. Western blot analysis (anti-NSP5) of soluble and insoluble fractions of untagged or N-terminally tagged wild-type and mutant NSP5, expressed in MA104 cells. Cells were lysed as described previously (2). Aliquots corresponding to 10% of supernatants from extracts centrifuged at 16,000 x g for 5 min at 4°C represented the soluble fraction (upper panels). The total pellet fraction was resuspended in 2% sodium dodecyl sulfate (insoluble fraction) and loaded (lower panels). A, fractions from cells transfected with the indicated NSP5 constructs. B, fractions from cells expressing NSP5 alone or with NSP2. C, fractions from SA11 virus-infected cells. Open and closed arrowheads indicate the nonphosphorylated (26 kDa) and the phosphorylated (28 kDa and higher) bands, respectively.

Sen et al. further claim that NSP5 phosphorylation is independent of NSP2. We addressed this point as well by analyzing NSP5 phosphorylation when coexpressed with NSP2, both in the soluble and insoluble fractions. We found, in agreement with our previous results (1), that NSP2 was able to induce hyperphosphorylation of wtNSP5, with identical forms being present in the insoluble fraction but with the amount representing less than 10% (Fig. 1B). Finally, and very importantly, at least 90% of the total amount of wtNSP5 derived from virus-infected cells was present in the soluble fraction (Fig. 1C). Therefore, we can rule out the possibility that lack of analysis of the insoluble fractions could have led to misinterpretation of our previously published data.

Our results clearly show that without the control of the natural protein, the use of tagged-NSP5 constructs can produce spurious and misleading results. Daring statements on biochemical properties of NSP5 seem to be premature when the data on which they are based are derived only from proteins modified by the addition of tags.


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REFERENCES
 
    1
  1. Afrikanova, I., E. Fabbretti, M. C. Miozzo, and O. R. Burrone. 1998. Rotavirus NSP5 phosphorylation is up-regulated by interaction with NSP2. J. Gen. Virol. 79:2679-2686.[Abstract]
    2. 2
  2. Afrikanova, I., M. C. Miozzo, S. Giambiagi, and O. Burrone. 1996. Phosphorylation generates different forms of rotavirus NSP5. J. Gen. Virol. 77:2059-2065.[Abstract/Free Full Text]
    3. 3
  3. Eichwald, C., G. Jacob, B. Muszynski, J. E. Allende, and O. R. Burrone. 2004. Uncoupling substrate and activation functions of rotavirus NSP5: phosphorylation of Ser-67 by casein kinase 1 is essential for hyperphosphorylation. Proc. Natl. Acad. Sci. USA 101:16304-16309.[Abstract/Free Full Text]
    4. 4
  4. Mohan, K. V., J. Muller, and C. D. Atreya. 2003. The N- and C-terminal regions of rotavirus NSP5 are the critical determinants for the formation of viroplasm-like structures independent of NSP2. J. Virol. 77:12184-12192.[Abstract/Free Full Text]
    5. 5
  5. Sen, A., D. Agresti, and E. R. Mackow. 2006. Hyperphosphorylation of the rotavirus NSP5 protein is independent of serine 67 or NSP2, and the intrinsic insolubility of NSP5 is regulated by cellular phosphatases. J. Virol. 80:1807-1816. (Erratum, 80:3692.)[Free Full Text]
Michela Campagna
Oscar R. Burrone*
International Centre for Genetic Engineering and Biotechnology
Area Science Park, Padriciano 99
34012 Trieste, Italy,1

* Phone: 3904037571, Fax: 39040226555, E-mail: burrone{at}icgeb.org


Authors' Reply


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LETTER 
 
Our studies verify several reports on the hyperphosphorylation of native and tagged NSP5 (3, 4, 14, 15, 17) and address the controversy between findings of the Burrone group and those of other rotavirus investigators (1-9, 14-17). We and others have demonstrated that NSP5 is hyperphosphorylated in the absence of NSP2, regardless of the presence or position of tags, and that hyperphosphorylation is regulated by cellular phosphatases (3, 4, 14-17).

Native NSP5 findings are consistent with those for N-tagged NSP5. Prior reports on NSP5 hyperphosphorylation agree with our data on tagged NSP5 phosphorylation (3, 4, 14-17). Previously reported findings include the following: (i) the hyperphosphorylation of untagged NSP5 in the absence of NSP2 (3, 4); (ii) phosphatase regulation of untagged NSP5 or NSP5 made during rotavirus infection (4); (iii) the specific association of untagged NSP5 with N-tagged NSP5 (14); and (iv) the ability of NSP5 alone to form viroplasm-like structures (VLS) (14). Our findings using N- and C-terminally tagged NSP5 confirm and extend these reports using untagged NSP5 and suggest that the tagged versions employed are not spurious but are representative of findings with native NSP5 proteins.

Kinases and phosphatases regulate NSP5 hyperphosphorylation. The constitutive dephosphorylation of NSP5 by cellular phosphatases is a fundamental determinant of NSP5 hyperphosphorylation (4, 16). Of note, the phosphatase regulation of NSP5 does not appear to have been considered in Dr. Burrone's reply or model of NSP5 hyperphosphorylation (1, 2, 5-9). When soluble and insoluble fractions are delineated using internal cell protein markers, we have shown that hyperphosphorylated NSP5 isoforms are primarily present in the insoluble fraction (>80%) (Fig. 1) (16). The accumulation of soluble hyperphosphorylated NSP5 isoforms occurs only when phosphatases are inhibited (4, 16). As a result, NSP5 hyperphosphorylation is the sum of kinase and phosphatase activity on NSP5, and the level of hyperphosphorylated NSP5 is altered simply by decreasing phosphatase activity. NSP2 interacts with the N terminus of NSP5 (1, 8) but may function by blocking or reducing the dephosphorylation of soluble NSP5 without altering NSP5 phosphorylation.

NSP5 model of phosphorylation is based on N-tagged NSP5 constructs. The Burrone model of NSP5 phosphorylation is itself based on the use of N-terminally tagged NSP5 proteins and deletion mutants (5). Therefore, the reported model of NSP5 hyperphosphorylation is similarly subject to scrutiny and criticism for having used N-tagged NSP5 constructs, and the statements at the end of the reply seem equally applicable to the N-tagged NSP5 data used to develop their model.

Further, the reply and model use vaccinia virus infection in the evaluation of NSP5 hyperphosphorylation (1, 2, 5-9). However, vaccinia virus encodes several kinases and phosphatases that regulate cell signaling pathways and dramatically affect cellular phosphorylation events in addition to altering cytoskeletal associations (11-13). The role of vaccinia virus kinases and phosphatases need to be considered when evaluating "native" mechanisms of NSP5 hyperphosphorylation.

Analysis of basal versus hyperphosphorylated forms of NSP5. The reply data do not address the hyperphosphorylation of untagged NSP5. There are two basal isoforms of NSP5, of 26 and 28 kDa, the latter of which is O glycosylated to account for its decreased mobility (9). Hyperphosphorylation of NSP5 reportedly results in the generation of a number of bands in the 32- to 35-kDa range (3, 4, 15). However, in the reply data, the higher basal isoform is indicated as that of hyperphosphorylated NSP5. No further analysis of hyperphosphorylated 32- to 35-kDa NSP5 isoforms is presented, and no hyperphosphorylated isoforms are apparent with or without NSP2 or serine 67. Since the reply data lack molecular markers and there are no side-by-side comparisons of the rotavirus-expressed NSP5 with the vaccinia virus T7-expressed NSP5, it is unclear whether hyperphosphorylated NSP5 isoforms were analyzed. The figures also appear to lack controls for protein loading, soluble and insoluble fraction authenticity, and comparable sample separation. It is also unclear why >10-fold-lower levels of untagged NSP5 are compared to tagged NSP5 and whether relevant conclusions can be drawn from this comparison.

Data do not reproduce or establish solubility/insolubility parameters. The reply does not use comparable methods for separating soluble and insoluble fractions as reported by us and others, nor are data validated by the analysis of insoluble cellular proteins within samples (10, 16). The low-speed short centrifugation used in the reply is inadequate for separating soluble from insoluble fractions, and claims about the percentage of NSP5 within soluble or insoluble fractions cannot be determined without establishing these parameters. Curiously, two identical untagged NSP5 samples in the reply provide completely different solubility and insolubility percentages.

The reply data do in fact confirm that insoluble hyperphosphorylated NSP5 is present in rotavirus-infected cells. This validates the Magnussen group's paper, which documented the presence of insoluble NSP5 during rotavirus infection (4), and further questions the assertion that insoluble NSP5 protein is unimportant.

Recent findings further support our conclusions. When a 30-min 20,000 x g centrifugation is employed, we find that >80% of NSP5 is present in the insoluble fraction and that NSP5 is hyperphosphorylated without NSP2 (Fig. 1). The soluble fraction contains basal isoforms of NSP5, while hyperphosphorylated 32- to 35-kDa isoforms of NSP5 are present in the insoluble fraction. This is similar to hyperphosphorylated isoforms reported following rotavirus infection or following expression of untagged NSP5 (3, 4, 15).

The reply data demonstrate that the NSP5 S67A mutation results in hyperphosphorylation, confirming our findings (16). However, we have recently found that mutagenesis of a single amino acid (MutA) at the C terminus of NSP5 completely disrupts hyperphosphorylation of the N-tagged NSP5 (Fig. 1). These data demonstrate that changing a single residue determines NSP5 hyperphosphorylation and that hyperphosphorylation of N-tagged NSP5 is quite specific.

It is clear that either tagged or untagged proteins can result in spurious or misleading results and that rigorous testing of underlying assumptions, systems, and data will be required to validate proposed models.


Figure 2
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  		FIG. 1. Single amino acid mutation results in complete loss of N-tagged NSP5 (N-NSP5) hyperphosphorylation. A single amino acid mutation (MUT-A) was introduced in the N-NSP5 carboxyl-terminal dimerization region proposed to be critical for hyperphosphorylation (5, 17). COS-7 cells were transfected with either wild-type or Mut-A NSP5 plasmids, and 48 h posttransfection, NSP5 was analyzed in 1% NP-40 lysis buffer. Equivalent amounts of soluble and insoluble fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting of 6-His N-terminal tags (16). More than 80% of wild-type NSP5 is found in the insoluble fraction, within basal and hyperphosphorylated isoforms. Soluble fractions contain only basal isoforms of NSP5, in the absence of hyperphosphorylated isoforms (16). Interestingly, the Mut-A NSP5 lacks hyperphosphorylated isoforms and accumulates primarily in the soluble fraction in basal isoforms. *, a cellular band at the top of the gel provides an internal control for comparable loading and separation of samples.


arrow
REFERENCES 
 
    1
  1. Afrikanova, I., E. Fabbretti, M. C. Miozzo, and O. R. Burrone. 1998. Rotavirus NSP5 phosphorylation is up-regulated by interaction with NSP2. J. Gen. Virol. 79:2679-2686.[Abstract]
    2. 2
  2. Afrikanova, I., M. C. Miozzo, S. Giambiagi, and O. Burrone. 1996. Phosphorylation generates different forms of rotavirus NSP5. J. Gen. Virol. 77:2059-2065.[Abstract/Free Full Text]
    3. 3
  3. Blackhall, J., A. Fuentes, K. Hansen, and G. Magnusson. 1997. Serine protein kinase activity associated with rotavirus phosphoprotein NSP5. J. Virol. 71:138-144.[Abstract]
    4. 4
  4. Blackhall, J., M. Munoz, A. Fuentes, and G. Magnusson. 1998. Analysis of rotavirus nonstructural protein NSP5 phosphorylation. J. Virol. 72:6398-6405.[Abstract/Free Full Text]
    5. 5
  5. Eichwald, C., G. Jacob, B. Muszynski, J. E. Allende, and O. R. Burrone. 2004. Uncoupling substrate and activation functions of rotavirus NSP5: phosphorylation of Ser-67 by casein kinase 1 is essential for hyperphosphorylation. Proc. Natl. Acad. Sci. USA 101:16304-16309.[Abstract/Free Full Text]
    6. 6
  6. Eichwald, C., J. F. Rodriguez, and O. R. Burrone. 2004. Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation. J. Gen. Virol. 85:625-634.[Abstract/Free Full Text]
    7. 7
  7. Eichwald, C., F. Vascotto, E. Fabbretti, and O. R. Burrone. 2002. Rotavirus NSP5: mapping phosphorylation sites and kinase activation and viroplasm localization domains. J. Virol. 76:3461-3470.[Abstract/Free Full Text]
    8. 8
  8. Fabbretti, E., I. Afrikanova, F. Vascotto, and O. R. Burrone. 1999. Two non-structural rotavirus proteins, NSP2 and NSP5, form viroplasm-like structures in vivo. J. Gen. Virol. 80:333-339.[Abstract]
    9. 9
  9. Gonzalez, S. A., and O. R. Burrone. 1991. Rotavirus NS26 is modified by addition of single O-linked residues of N-acetylglucosamine. Virology 182:8-16.[CrossRef][Medline]
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  10. Harlow, E., and D. Lane. 1988. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    11. 11
  11. Hollinshead, M., G. Rodger, H. V. Eijl, M. Law, R. Hollinshead, D. J. T. Vaux, and G. L. Smith. 2001. Vaccinia virus utilizes microtubules for movement to the cell surface. J. Cell Biol. 154:389-402.[Abstract/Free Full Text]
    12. 12
  12. Liu, K., B. Lemon, and P. Traktman. 1995. The dual-specificity phosphatase encoded by vaccinia virus, VH1, is essential for viral transcription in vivo and in vitro. J. Virol. 69:7823-7834.[Abstract]
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  13. Lin, S., and S. S. Broyles. 1994. Vaccinia protein kinase 2: a second essential serine/threonine protein kinase encoded by vaccinia virus. Proc. Natl. Acad. Sci. USA 91:7653-7657.[Abstract/Free Full Text]
    14. 14
  14. Mohan, K. V., J. Muller, I. Som, and C. D. Atreya. 2003. The N- and C-terminal regions of rotavirus NSP5 are the critical determinants for the formation of viroplasm-like structures independent of NSP2. J. Virol. 77:12184-12192.[Abstract/Free Full Text]
    15. 15
  15. Poncet, D., P. Lindenbaum, R. L'Haridon, and J. Cohen. 1997. In vivo and in vitro phosphorylation of rotavirus NSP5 correlates with its localization in viroplasms. J. Virol. 71:34-41.[Abstract]
    16. 16
  16. Sen, A., D. Agresti, and E. R. Mackow. 2006. Hyperphosphorylation of the rotavirus NSP5 protein is independent of serine 67 or NSP2, and the intrinsic insolubility of NSP5 is regulated by cellular phosphatases. J. Virol. 80:1807-1816. (Erratum, 80:3692.)[Free Full Text]
    17. 17
  17. Torres-Vega, M. A., R. A. Gonzalez, M. Duarte, D. Poncet, S. Lopez, and C. F. Arias. 2000. The C-terminal domain of rotavirus NSP5 is essential for its multimerization, hyperphosphorylation and interaction with NSP6. J. Gen. Virol. 81:821-830.[Abstract/Free Full Text]
Adrish Sen
Erich R. Mackow*
Stony Brook University
Department of Medicine and Molecular Genetics and Microbiology
HSC T17, Rm. 48
Stony Brook, NY 11794-8173,2

* Phone: (631) 444-2120, Fax: (631) 444-8886, E-mail: emackow{at}mail.som.sunysb.edu

Journal of Virology, August 2006, p. 8283-8285, Vol. 80, No. 16
0022-538X/06/$08.00+0     doi:10.1128/JVI.00813-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Sen, A., Sen, N., Mackow, E. R. (2007). The Formation of Viroplasm-Like Structures by the Rotavirus NSP5 Protein Is Calcium Regulated and Directed by a C-Terminal Helical Domain. J. Virol. 81: 11758-11767 [Abstract] [Full Text]  
  • Campagna, M., Budini, M., Arnoldi, F., Desselberger, U., Allende, J. E., Burrone, O. R. (2007). Impaired hyperphosphorylation of rotavirus NSP5 in cells depleted of casein kinase 1{alpha} is associated with the formation of viroplasms with altered morphology and a moderate decrease in virus replication. J. Gen. Virol. 88: 2800-2810 [Abstract] [Full Text]  
  • Arnoldi, F., Campagna, M., Eichwald, C., Desselberger, U., Burrone, O. R. (2007). Interaction of Rotavirus Polymerase VP1 with Nonstructural Protein NSP5 Is Stronger than That with NSP2. J. Virol. 81: 2128-2137 [Abstract] [Full Text]  

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